Six-plex MS/MS method to measure enzyme activities related to MPS II, MPS IIIB, MPS IVA, MPS VI, MPS VII and CLN2 disorders in a single dried blood spot Jonathan Rehnberg; PerkinElmer, Turku, Finland FI-20101 Anna Potier, Jason Cournoyer, Joe Trometer, and Mack Schermer; PerkinElmer, Waltham, MA 02451 Alyssa Vranish and Jim DiPerna; PerkinElmer, Bridgeville, PA 15017 Yang Liu, Fan Yi, Naveen Kumar Chennamaneni, Zdenek Spacil, Arun Babu Kumar, Joyce Liao, Michael H. Gelb, C. Ronald Scott and Frantisek Turecek; University of Washington, Seattle, WA 98195 Introduction Methods 1 3 Defects in the metabolic breakdown of cellular debris due to deficient activity of lysosomal enzymes can result in a lysosomal storage disorder (LSD). For example, deficient activity of the lysosomal enzyme GALNS causes accumulation of glycosaminoglycans (GAGs) that causes MPS IVA disease, also known as Morquio A disease. We show here that deficient activity of lysosomal enzymes related to 6 different LSDs can be detected from a single 3.2 mm newborn dried blood spot (DBS) simultaneously using mass spectrometry. The six disorders (and enzymes) are MPS II (I2S), MPS IIIB (NAGLU), MPS IVA (GALNS), MPS VI (ARSB), MPS VII (GUSB) and CLN2 (TPP1). Briefly, DBS are incubated overnight at 37°C in a cocktail containing enzyme specific substrates and internal standards (IS) followed by a post-incubation workup that is less than 30 minutes per 96-well plate. With further optimization the sample-to-sample analysis time can be as low as 2 minutes, which allows the possibility to obtain more than ~4300 results per day if desired. Method performance studies show good day-to-day reproducibility for controls and excellent differentiation of a DBS from individuals with confirmed low I2S/NAGLU/GALNS/ARSB/GUSB/TPP1 activity from a cohort of DBS from several hundred presumed healthy neonates. Samples were analyzed using a Waters Xevo TQD with an X-select CSH C18 column and a Vanguard CSH precolumn. A flow solvent consisting of 60:40 H2O:Acetonitrile with 0.1% formic acid at 0.5 mL/min showed good separation between the internal standard and residual substrate peaks for each analyte. To assess assay performance, a study consisting of DBS from de-identified presumed healthy subjects (N=616) collected in August 2016 and DBS with confirmed low (N=29) activity for I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 were run over 3 days (~3 plates per day). Each plate had 2 blanks (punched blank filter paper) and two replicates of each control level. Assay precision was estimated using the 18 control replicates each of low/mid/high over the 3 days. Materials and Assay Procedure 2 The assay procedure is detailed in the workflow diagram in Figure 1. The formulation of the incubation cocktail is in Table 1 and the structures of the substrates and IS are in Figure 2. Punch Shake & Incubate Add Cocktail Quench & Mix Transfer Liquid & Extract Figure 3. Dot plots for the enzymes I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 measured in DBS from the presumptive normal neonatal, DBS from individuals with confirmed low enzyme activity and low, mid and high controls. Add 30 µL cocktail Seal plate 37°C, 400 rpm 18 ± 2 hours Remove seal, add 100 µL 50:50 MeOH:EtOAc Mix with pipette Transfer to 96 deep well plate (DWP) Add 400 µL EtOAc then 200 µL 0.5 M NaCl Solution Mix with pipette 1 2 3 4 3.2 mm DBS punch to 96-well plate Results 4 Transfer Top Layer Shake Add Flow Solvent Dry Separate Layers Dot plots of the results for the six enzymes are shown in Figure 3. Multiple replicates (N=18) of the low, mid and high control DBS showed good precision in the assay for all six enzymes. Table 2 shows that all controls had %CV ≤ 11% except the low control for NAGLU and TPP1. The data shows clear distinction between the presumptive normal and the confirmed low activity DBS suggesting that a cut off can easily be made to routinely differentiate low activity from normal neonatal DBS. Furthermore, all confirmed low activity samples were less than 10% of the average presumptive neonatal population and also less than the lowest presumptive normal activity for each enzyme, as shown in Table 2. MSMS Room temp (25°C) at 400 rpm 10 min Reconstitute with 100 µL 60:40 H2O:ACN with 0.1% formic acid Evaporate at 40°C, 10-15 min Transfer 200 µL top layer to sampling plate Centrifuge DWP 5 min, 700 x g 10uL injection using X-Select column 9 8 7 6 5 Figure 1. 6-plex Assay Protocol Formulation of 96 sample S+IS vial (3.3 mL) assay cocktail Substrates MW µM mg/vial Int. Standards µg/vial I2S-S 723.3 500 1.19 I2S-IS 648.3 5 11 NAGLU-S 622.4 1.03 NAGLU-IS 422.3 7 GALNS-S 764.3 1000 2.52 GALNS-IS 689.4 ARSB-S 736.3 2.42 ARSB-IS 661.4 GUSB-S 609.3 1.01 GUSB-IS 440.3 10 15 TPP1-S 638.4 200 0.42 TPP1-IS 358.3 18 Buffer Composition Reagents mM Ammonium Acetate (pH 5.0) 77.1 60 Cerium Acetate 317.3 NAG-thiazoline 219.3 0.1 Figure 1. 6-plex Assay Protocol Type Enzyme Activity (mM/hr) I2S NAGLU GALNS ARSB GUSB TPP1 Low Control 0.78 0.21 0.46 1.68 8.51 5.33 %CV 7% 24% 9% 10% 11% 22% Mid Control 6.89 2.68 3.04 11.6 25.7 19.9 5% 6% 8% High Control 13.0 5.85 6.14 24.3 45.1 24.9 Average Normal Neonatal 13.8 2.56 2.52 11.0 27.7 20.4 Lowest Normal Neonatal 6.74 1.17 0.72 3.33 14.8 Highest Confirmed Low 1.22 0.24 1.13 0.11 0.64 Table 1. 6-plex incubation cocktail components Substrates Internal Standards Table 2. Average activities for control DBS and DBS from presumptive normal neonatal DBS for the enzymes I2S, NAGLU, GALNS, ARSB, GUSB and TPP1. The %CV for the controls is based on 18 replicates over 3 days. The lowest normal neonatal is the lowest activity measured of the 616 DBS analyzed and the highest confirmed low is the highest measured activity in the confirmed low-activity cohort. I2S NAGLU GALNS Summary 5 A new tandem mass spectrometric multiplex assay for measuring I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 enzyme activities in DBS was demonstrated. This method was able to clearly differentiate between the activities in samples collected from presumed healthy subjects and in DBS samples from individuals confirmed to have low enzyme activity. Good precision of each method was demonstrated using control DBS. We thank the Mayo Clinic, Greenwood Genetics and Serv. Genet. Med. HCPA for providing confirmed low activity DBS samples. All DBS samples were collected following IRB approved protocols. ARSB GUSB TPP1 Figure 2. Structures of 6-plex substrates and IS